Biology subjects

Toro, E.

Publications and source records attributed to Toro, E..

4 recordsLinked to original sources

bioRxiv2024

High-throughput DNA engineering by mating bacteria

To reduce the operational friction and scale DNA engineering, we report here an in vivo DNA assembly technology platform called SCRIVENER (Sequential Conjugation and Recombination for In Vivo Elongation of Nucleotides with low ERrors). SCRIVENER combines bacterial conjugation, in vivo DNA cutting, and in vivo homologous recombination to seamlessly stitch blocks of DNA together by mating E. coli in large arrays or pools. This workflow is simpler, cheaper, and higher throughput than current DNA assembly approaches that require DNA to be moved in and out of cells at different procedural steps. We perform over 5,000 assemblies with two to 13 DNA blocks that range from 240 bp to 8 kb and show that SCRIVENER is capable of assembling constructs as long as 23 kb at relatively high throughput and fidelity. Most SCRIVENER errors are deletions between long interspersed repeats. However, SCRIVENER can overcome these errors by enabling assembly and sequence verification at high replication at a nominal additional cost per replicate. We show that SCRIVENER can be used to build combinatorial libraries in arrays or pools, and that DNA blocks onboarded into the platform can be repurposed and reused with any other DNA block in high throughput without a PCR step. Because of these features, DNA engineering with SCRIVENER has the potential to accelerate design-build-test-learn cycles of DNA products.

synthetic biology↗

bioRxiv2020

Systematic genome-wide querying of coding and non-coding functional elements in E. coli using CRISPRi

Genome-wide repression screens using CRISPR interference (CRISPRi) have enabled the high-throughput identification of essential genes in bacteria. However, there is a lack of functional studies leveraging CRISPRi to systematically explore targeting of both the coding and non-coding genome in bacteria. Here we perform CRISPRi screens in Escherichia coli MG1655 K-12 targeting ~13,000 genomic features, including nearly all protein-coding genes, non-coding RNAs, promoters, and transcription factor binding sites (TFBSs) using a ~33,000-member sgRNA library, which represents the most compact and comprehensive genome-wide CRISPRi library in E. coli to date. Our data reveal insights into the conditional essentiality of the genome with key refinements to screen design and profiling. First, we demonstrate that strong fitness defects associated with essential cellular processes can be resolved using inducible time-series measurements. We show that knockdowns of different classes of genes exhibit distinct, transient responses that are correlated to gene function with genes involved in translation exhibiting the strongest responses. We also query feature essentiality across several biochemical conditions and show that several genes, sRNAs, and operons exhibit conditional phenotypes not reported by previous high-throughput efforts. Second, we evaluate systematically targeting non-genic features (promoters and TFBSs) in the E. coli genome. We show that promoter-targeting guides can be used to add phenotypic confidence to promoter annotations and verify computationally predicted promoters. In contrast to prior studies, we find that promoter knockdowns exhibit a strong targeting orientation dependency where targeting the non-template strand of the promoter closest to the target gene is more effective in knocking down gene expression than other promoter targeting orientations. Unlike eukaryotic genomes, we note that interpreting the effects of TFBS targeting is particularly challenging due to the small size of such features and their proximity to and overlap with other genomic features. Together, this work reveals novel conditionally essential gene phenotypes, provides a characterized set of sgRNAs for future E. coli CRISPRi screens, and highlights considerations for CRISPRi library design and screening for microbial genome characterization.

microbiology↗

bioRxiv2018

A versatile platform strain for high-fidelity multiplex genome editing

Precision genome editing accelerates the discovery of the genetic determinants of phenotype and the engineering of novel behaviors in organisms. Advances in DNA synthesis and recombineering have enabled high-throughput engineering of genetic circuits and biosynthetic pathways via directed mutagenesis of bacterial chromosomes. However, the highest recombination efficiencies have to date been reported in persistent mutator strains, which suffer from reduced genomic fidelity. The absence of inducible transcriptional regulators in these strains also prevents concurrent control of genome engineering tools and engineered functions. Here, we introduce a new recombineering platform strain, BioDesignER, which incorporates (1) a refactored {lambda}-Red recombination system that reduces toxicity and accelerates multi-cycle recombination, (2) genetic modifications that boost recombination efficiency, and (3) four independent inducible regulators to control engineered functions. These modifications resulted in single-cycle recombineering efficiencies of up to 25% with a seven-fold increase in recombineering fidelity compared to the widely used recombineering strain EcNR2. To facilitate genome engineering in BioDesignER, we have curated eight context-neutral genomic loci, termed Safe Sites, for stable gene expression and consistent recombination efficiency. BioDesignER is a platform to develop and optimize engineered cellular functions and can serve as a model to implement comparable recombination and regulatory systems in other bacteria.

synthetic biology↗

bioRxiv2018

Intracranial fluid dynamics changes in idiopathic intracranial hypertension: pre and post therapy

Idiopathic intracranial hypertension (IIH) is a condition of unknown etiology frequently associated with dural sinus stenosis. There is emerging evidence that venous sinus stenting is an effective treatment. We use phase contrast cine MRI to observe changes in flow dynamics of multiple intracranial fluids and their response to different treatments in a patient with IIH. We quantified the following parameters at the level of the aqueduct of Sylvius and the cervical C2C3: cerebrospinal fluid (CSF), arterial and venous flow; CSF velocity amplitude; artero-venous delay (AVD); artero-CSF delay and percentage of venous outflow normalized to total arterial inflow (tIJV/tA). Analyses were run before lumbar puncture (LP) (A), after LP (B), after medical therapy (C) and after venous stent placements deployed at two separate times (D and E). AVD and tIJV/tA improved only after CSF removal and after stent placements. CSF velocity amplitude remained elevated. Arterial flow profile showed a dramatic reduction after LP with improvement in mean venous flow. This report is the first to demonstrate interactive changes in intracranial fluid dynamics that occur before and after different therapeutic interventions in IIH. We discuss how increased intracranial venous blood could be \"tumoral\" in IIH and facilitating its outflow could be therapeutic.

neuroscience↗